Heterogeneity of colorectal cancer (CRC) in reference to KRAS proto-oncogene utilizing WAVE technology.

BACKGROUND: New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10-20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS. METHODS: In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by the Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (high performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for the presence of KRAS proto-oncogene mutations at codons 12 and 13. RESULTS: Initially, the sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by direct sequencing. All four samples from patient 2 tested positive for Exon 12/13 mutations. Of the seven samples from patient 3, five were positive for Exon 12/13 mutations and two were negative for Exon 12/13 mutations. CONCLUSION: In these studies the analysis of three patients' colorectal cancer tissues were analyzed utilizing the WAVE technology. Results demonstrated a greater degree of sensitivity in mutation detection when compared to standard sequencing. These studies also demonstrated heterogeneity of expression of KRAS mutations between areas of the tissue samples at a genomic level. The low clinical response rates to EGFR inhibition might be explained by the variation in mutation presence, which was dependent upon the region examined. The heterogeneity demonstrated in these studies provides another phenotypic variant that will impact clinical care.

Metastatic malignant melanoma presenting as an appendiceal mucocele.

Melanoma metastatic to the appendix is extremely rare. Here we describe a case of a 31-year-old female from Bolivia with a remote history of metastatic malignant melanoma first diagnosed as a cutaneous malignant melanoma ten years prior to this presentation. The patient was being followed for a mucocele which on resection was found to be metastatic melanoma. "Mucocele" is a generic diagnosis that warrants further characterization and treatment.

Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle.

The contraction of gallbladders (GBs) with cholesterol stones is impaired due to high cholesterol concentrations in caveolae compared with GBs with pigment stones. The reduced contraction is caused by a lower cholecystokinin (CCK)-8 binding to CCK-1 receptors (CCK-1R) due to caveolar sequestration of receptors. We aimed to examine the mechanism of cholesterol-induced sequestration of receptors. Muscle cells from human and guinea pig GBs were studied. Antibodies were used to examine CCK-1R, antigens of early and recycling endosomes, and total (CAV-3) and phosphorylated caveolar-3 protein (pCAV-3) by Western blots. Contraction was measured in muscle cells transfected with CAV3 mRNA or clathrin heavy-chain small-interfering RNA (siRNA). CCK-1R returned back to the bulk plasma membrane (PM) 30 min after CCK-8 recycled by endosomes, peaking at 5 min in early endosomes and at 20 min in recycling endosomes. Pretreatment with cholesterol-rich liposomes inhibited the transfer of CCK-1R and of CAV-3 in the endosomes by blocking CAV-3 phosphorylation. 4-Amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (inhibitor of tyrosine kinase) reproduced these effects by blocking pCAV-3 formation, increasing CAV-3 and CCK-1R sequestration in the caveolae and impairing CCK-8-induced contraction. CAV-3 siRNA reduced CAV-3 protein expression, decreased CCK-8-induced contraction, and accumulated CCK-1R in the caveolae. Abnormal concentrations of caveolar cholesterol had no effect on met-enkephalin that stimulates a delta-opioid receptor that internalizes through clathrin. We found that impaired muscle contraction in GBs with cholesterol stones is due to high caveolar levels of cholesterol that inhibits pCAV-3 generation. Caveolar cholesterol increases the caveolar sequestration of CAV-3 and CCK-1R caused by their reduced recycling to the PM.

Does sodium hyaluronate- and carboxymethylcellulose-based bioresorbable membrane (Seprafilm) decrease operative time for loop ileostomy closure?

BACKGROUND: Adhesions can result in serious clinical complications and make ileostomy closure, which is relatively simple procedure into a complicated and prolonged one. The use of sodium hyaluronate and carboxymethyl cellulose membrane (Seprafilm) was proven to significantly reduce the postoperative adhesions at the site of application. The aim of this study was to assess the incidence and severity of adhesions around a loop ileostomy and to analyze the length of time and morbidity for mobilization at the time of ileostomy closure with and without the use of Seprafilm. METHODS: Twenty-nine surgeons from 15 institutions participated in this multicenter prospective randomized study. 191 patients with loop ileostomy construction were randomly assigned to either receive Seprafilm under the midline incision and around the stoma (Group I), only under the midline incision (Group II), or not to receive Seprafilm (Group III). At ileostomy closure, adhesions were quantified and graded; operative morbidity was also measured. RESULTS: All 3 groups were comparable relative to gender, mean age and number of patients with prior operations (26, 25 and 19, respectively). Group II patients were significantly more likely to have pre-existing adhesions than Group III patients (30.6% vs. 14.1%, p = 0.025). At stoma mobilization, significantly more patients in Group III than in Group I had adhesions around the stoma (95.2% vs. 82.3%, p = 0.021). Mean operative times were 27, 25, and 28 minutes, respectively (p = 0.38), with significant differences among sites. There was no significant difference in the number of patients needing myotomy or enterotomy (29, 27 and 24 patients, respectively), nor in the number of postoperative complications (7, 9 and 7 patients, respectively). CONCLUSIONS: When consistently applied, Seprafilm significantly decreased adhesion formation around the stoma but not operative times without any increase in the need for myotomy or enterotomy. These findings were not seen in the overall study population possibly due to the large number of surgeons using a variety of application techniques.

Gq-linked NK(2) receptors mediate neurally induced contraction of human sigmoid circular smooth muscle.

BACKGROUND & AIMS: Because tachykinins have been identified as neurotransmitters in the guinea pig colon and human ileum, we examined a possible role of tachykinin receptors and neurokinin (NK) A in neurally induced contraction of human sigmoid colon circular muscle. METHODS: Muscle strips were stimulated electrically for 10 seconds. Single cells were isolated by enzymatic digestion and permeabilized by saponin. [(35)S]GTPgammaS binding was assayed with or without NKA for 5 minutes. Intracellular Ca(2+) was measured using Fura 2. RESULTS: In the presence of 100 micromol/L L-NNA, 100 micromol/L atropine did not affect electrical field stimulation (EFS)-induced contraction. A peptide NK(2)-receptor antagonist (NK-2ra) but not an NK(1) antagonist FK888 (1 micromol/L) eliminated EFS-induced contraction. NKA-induced contraction in muscle strips and single cells was virtually abolished by NK-2ra, but not by FK888. In permeabilized cells, contraction was blocked by Gq-protein antibodies, but not by other G-protein antibodies, suggesting that NKA activates Gq, which was confirmed by a [(35)S]GTPgammaS binding assay. NKA-induced contraction and increase in cytosolic Ca(2+) were abolished by depletion of intracellular Ca(2+) stores. CONCLUSIONS: Tachykinins may be the main excitatory neurotransmitters in human sigmoid circular muscle. NKA activates Gq-linked NK(2) receptors, which cause Ca(2+) release, followed by contraction.

Colostomy closure: impact of preoperative risk factors on morbidity.

The objective of this study was to stratify patients for colostomy closure into risk categories according to preoperative variables. This was a retrospective case series. Median follow-up was 82 months. A tertiary care academic medical center was the setting for this study. A study sample of 155 consecutive patients who underwent colostomy closure at a single institution between 1985 and 1995 were included in this study. The following preoperative variables were analyzed: indication for colostomy fashioning; age; gender; American Society of Anesthesiology (ASA) class; presence of cardiac, renal, or pulmonary dysfunctions; presence of diabetes mellitus; and immunosuppression. The occurrence of adverse outcome, as evidenced by postoperative morbidity and mortality, was used as the main outcome measure. Complications occurred in 49 patients (31.6%), including a 1.3 per cent mortality. There was a trend of increasing morbidity with increasing ASA class. The single factor that showed a statistically significant increase in morbidity was the presence of diabetes (P = 0.036). Predicted probabilities of complications for patients with ASA III with renal disease was 31 per cent, increased to 47.9 per cent if cardiac disease was also present and to 77 per cent with the addition of diabetes. The presence of diabetes carries an independent risk factor for adverse outcome in colostomy closure. This study provides information about stratification of postoperative risk based on commonly available preoperative variables. In the majority of cases, colostomy closure seems to carry a very acceptable complication rate. In selected patients with multiple preoperative risk factors, the morbidity becomes significantly higher.

Gastrointestinal malignancies in patients with celiac sprue.

BACKGROUND: Celiac sprue is a malabsorption disease, which carries an increased risk of gastrointestinal malignancy, often underestimated. The purpose of this study was to examine the management of patients with gastrointestinal neoplasms complicating celiac disease. PATIENTS AND METHODS: The pathology database at our institution was searched from 1986 to present; and the literature from 1966 to 1997 was reviewed to identify reports of celiac sprue complicated by malignancy. A total of 82 cases were available for analysis. RESULTS: Two thirds of patients had carried the diagnosis of celiac sprue for a mean of approximately 10 years. The remaining one third were diagnosed with celiac disease and gastrointestinal malignancy simultaneously. Jejunal T-cell lymphoma was the most common malignancy. There was also an increased frequency of small intestinal adenocarcinoma and squamous cell carcinoma of the esophagus. Prognosis was generally poor, related to the histologic type and stage of the disease. CONCLUSIONS: Gastrointestinal malignant neoplasms, especially small bowel lymphomas, can occur in patients with celiac sprue. Patients with known celiac disease who present with exacerbation of symptoms should be promptly investigated for occult gastrointestinal malignancies, and considered for early surgical exploration.

Mutated p53 gene is an independent adverse predictor of survival in colon carcinoma.

OBJECTIVE: To evaluate the impact of p53 gene mutations on long-term survival in patients with intermediate stage carcinoma of the colon. DESIGN: Retrospective cohort study; median follow-up of 87 months. SETTING: Tertiary care academic medical center. PATIENTS: Mutational analysis was conducted in a single institution in 141 consecutive patients with resected stage II (n = 71) and stage III (n = 70) colon carcinoma. Archival pathology specimens were analyzed for point mutations of exons from the p53 gene by means of amplification and direct sequencing by polymerase chain reaction. MAIN OUTCOME MEASURES: The impact of p53 mutations and of adverse histopathologic features (i.e., poor differentiation, lymphovascular invasion, or mucin production) on patient survival. RESULTS: Median overall survival was 64 months (95 months for patients with stage II and 34 months for patients with stage III colon carcinoma; P = .001). Presence of a p53 mutation was the single most important risk factor associated with poorer survival in both patients with stage II (P = .02) and stage III colon carcinoma (P = .006) throughout the follow-up period. A p53 mutation increased the risk of death by 2.82 times in patients with stage II and by 2.39 times in patients with stage III colon carcinoma. There was an additive effect on the cumulative risk of death between p53 mutations and adverse histopathologic variables. CONCLUSIONS: The presence of p53 mutations carries an independent adverse prognostic value in colon cancer. These findings imply that the applicability of mutational analysis in clinical practice is likely to affect therapeutic choices in the future.

Topographic genotyping of colorectal carcinoma: from a molecular carcinogenesis model to clinical relevance.

BACKGROUND: In recent years, as a result of refinement in molecular biology techniques, significant progress has been made in the understanding of colorectal carcinogenesis. Particular attention has been drawn to identification of genetic mutation that may predispose to colorectal carcinoma (familial syndromes) and may affect tumor behavior and prognosis (sporadic cases). CONCLUSIONS: Our method of topographic genotyping of human colonic carcinomas has shown a correlation between K-ras-2 and p53 mutations and stage at diagnosis as well as long-term survival. Data from other investigators in this field confirm the importance of genetic analysis of human colorectal tumors. These findings are likely to impact management by allowing a more individualized therapeutic approach.

Effects of lambda-carrageenan induced experimental enterocolitis on splenocyte function and nitric oxide production.

A lambda-carrageenan induced rat model of inflammatory bowel disease (IBD) allowed evaluation of progressive histopathological changes over time and comparison with human disease. In addition, this model was used to test the hypothesis that the immunosuppression observed in IBD may be mediated in part by augmented production of nitric oxide by splenic lymphocytes. Male Sprague-Dawley rats were fed standard rat chow and a drinking solution of 2% lambda-carrageenan without prior sensitization to the compound. At 2, 4, 6, and 8 weeks, small and large intestines were removed for histological examination, and splenocytes were assayed for response to various mitogens and for nitrite production. Intestinal lesions gradually developed in lambda-carrageenan fed rats and were found to be morphologically similar to those observed in human ulcerative colitis. The proliferative response of splenocytes to known mitogens was significantly diminished in carrageenan fed rats, when compared with pair-fed controls, but was fully restored when cultured in the presence of NG-monomethyl-L-arginine, a specific inhibitor of nitric oxide synthase. Preliminary data suggest that overproduction of nitric oxide may provide a molecular mechanism for the immunosuppression observed during chronic inflammatory bowel disease. Further characterization of a reproducible rat model of IBD will allow further investigation into the pathogenesis of the human disease.

Selective preservation of the anal transition zone in ileoanal pouch procedures.

PURPOSE: A prospective trial was conducted to evaluate use of certain preoperative criteria in the choice of operative technique for ileal pouch-anal anastomosis (IPAA). Handsewn vs. stapled anastomotic techniques were compared as was preservation vs. excision of the anal transition zone (ATZ). METHODS: Over an 18-month period, 40 consecutive patients underwent restorative proctocolectomy with IPAA for ulcerative colitis (31 cases) or familial adenomatous polyposis (9 cases). In 28 patients, ATZ was completely excised, by either a transanal mucosectomy with handsewn anastomosis (Group I, 13 cases) or by double-stapled technique (Group II, 15 cases). The ATZ was preserved and the anastomosis was double-stapled in colitis patients with suboptimum sphincter function and/or greater than 50 years of age in the absence of dysplasia or severe distal proctitis (Group III, 12 cases). RESULTS: Groups I and II patients were homogeneous in their preoperative variables and had equivalent functional outcome. Group III patients were older (P = 0.0001), with weaker preoperative anal sphincter resting tone (P = 0.024). Compared with Groups I and II, patients in Group III had significantly greater 24-hour stool frequency (P = 0.0056), daytime stool frequency (P = 0.0125), and incidence of daytime fecal seepage (P = 0.007). There was no significant difference in other outcome variables in Group III patients. There was no difference in morbidity in the three groups. CONCLUSIONS: Transanal mucosectomy with handsewn anastomosis provided early functional results equivalent to low anal transection with double-stapled IPAA in younger patients with excellent preoperative sphincter function. A double-stapled technique with preservation of the ATZ may be reserved for older patients, with poorer anal sphincter function, at minimum dysplasia/cancer risk, to optimize continence figures.

Peritoneal silicosis.

A 73-year-old man with a clinical diagnosis of pulmonary silicosis (long-standing exposure to silica, pulmonary infiltrates, and flu-like symptoms) presented to the emergency room with fever, acute biliary colic, and cholelithiasis. The patient had a 2-year status postchemotherapy with complete remission of hepatic and splenic malignant lymphoma. At laparotomy we found studding of the undersurface of the diaphragm with multiple small dark nodules. Owing to the patient's history of previously treated abdominal malignant lymphoma, the lesions were grossly interpreted as abdominal lymphomatosis. The microscopic appearance of the lesions suggested silicotic nodules, which were confirmed by digital scanning electron microscopy and roentgenographic microanalysis performed on formalin-fixed, paraffin-embedded tissue. This is an unusual extrapulmonary pattern of peritoneal seeding in silicosis.

Increased expression of keratinocyte growth factor messenger RNA associated with inflammatory bowel disease.

BACKGROUND & AIMS: Alterations in intestinal epithelial cell function are common in inflammatory bowel disease (IBD). Keratinocyte growth factor (KGF) is an epithelial cell-specific mitogen. The aim of this study was to examine IBD tissue for altered KGF expression. METHODS: Expression levels of the KGF and KGF receptor transcripts were analyzed by ribonuclease protection assay. The cellular localization of each transcript was determined using in situ hybridization. RESULTS: KGF messenger RNA levels were increased in inflamed IBD tissue in comparison with control tissues. In normal tissue, KGF messenger RNA was localized to the mesenchymal cells at the tip of the villi in the small intestine and directly underlying the mature enterocytes in the colon, whereas in IBD it was present throughout the lamina propria, although distinct from the germinal centers. The topographic distribution of the KGF in situ hybridization signal in IBD was similar to that observed for T lymphocytes. In contrast, KGF receptor transcripts were localized to the cryptal region of the mucosal epithelium in both normal and IBD tissue, with no apparent differences in the level of expression. CONCLUSIONS: The increased expression of KGF in IBD suggests that it may be involved in mediating the altered regulatory functions of intestinal epithelial cells in this disease.

Prognostic value of TP53 and K-ras-2 mutational analysis in stage III carcinoma of the colon.

BACKGROUND: Genetic mutations involving oncogenes and tumor-suppressor genes occur in carcinogenesis, and may affect biologic behavior of neoplasms. In this study, we analyzed the prognostic value of mutational analysis in colon carcinoma. PATIENTS AND METHODS: Archival pathology specimens from 70 consecutive patients, resected for stage III colon carcinoma, were analyzed for point mutations by amplification and direct sequencing of exons from the K-ras-2 and the TP53 genes (topographic genotyping). Mutations were compared with adverse histopathologic features (poor differentiation, vascular and lymphatic invasion, mucin production) as prognostic markers. RESULTS: Five-year survival was 75% in patients with nonmutated lesions, significantly lower (21%) with TP53 mutations (P = 0.01), and intermediate with K-ras-2 only (45%) or both K-ras-2 and TP53 mutations (36%). A TP53 mutation carried the highest relative risk of death (2.39; 95% confidence interval, 1.29 to 4.42; P = 0.006). There was an additive effect on the risk of death between TP53 mutations and adverse histopathologic features. CONCLUSIONS: The information derived from mutational analysis is creating new prognostic variables that may play a role in the choice of therapy for colorectal carcinoma.

Malignant potential in intestinal juvenile polyposis syndromes.

BACKGROUND: Unlike familial polyposis coli, where the premalignant nature of adenomatous polyps is well established, the cancer risk in juvenile polyposis has generally been considered not increased. METHODS: This study reviews all cases of juvenile polyposis reported in the English language to date to assess the occurrence and prognosis of carcinoma in the gastrointestinal tract. RESULTS: A total of 218 patients met the inclusion criteria. Mean age at diagnosis was 18.5 years (range: 9 months to 67 years). No gender preference was identified. The most common presenting symptom was chronic anemia, followed by acute gastrointestinal bleeding, rectal prolapse of polyp, protein-losing enteropathy, and intussusception. A family history of juvenile polyposis could be established in approximately 50% of patients, and associated congenital malformations were detected in 15%. Ninety-nine patients underwent 138 gastrointestinal operations: 121 colorectal, 12 gastric, and 5 small intestinal procedures. The development of a gastrointestinal carcinoma was reported in 36 cases (17%). Mean age at diagnosis of carcinoma was 35.5 years (range: 4-60 years). Most malignancies were located in the distal colon and rectum, with only one case of gastric and one case of duodenal carcinoma. Tumor stage at diagnosis was usually advanced, with poor survival figures. CONCLUSIONS: This study shows that juvenile polyposis syndromes carry a more significant risk of carcinoma than generally appreciated. Therefore, more intense endoscopic surveillance may be warranted, and definitive surgical options should often be considered in these syndromes.